Fluorescent molecules are widely used in numerous research and sensing applications. The scientific community in the field of the fluorescence detection is particularly interested in an understanding of how fluorescent molecules behave in various experimental geometries, especially in close proximity to metal and dielectric interfaces. In various previous studies, the radiation patterns of fluorescent molecules deposited on dielectric/dielectric or dielectric/metal interfaces have been calculated and observed. However, further development of the most efficient geometries for fluorescence detection is still needed.
Most previous Surface-Plasmon Resonance (SPR) sensors, excite the surface plasmons in a total internal reflection geometry. This is shown in Lakowicz, et al., U.S. Patent Application Publication 2005/0053974, where a sensing metal film is positioned on a high refractive index medium (glass), and the excitation light enters into the system for interaction with a metal film at an angle θ through the glass medium. In this arrangement, when a surface plasmon is excited, a significant enhancement of the optical field associated with the surface plasmon results. This evanescent field extends away from the metal film by about one wavelength of the illumination light beam into the air region next to the metal film. To fluoresce effectively, the fluorescent molecules must be oriented correctly and be localized in the vicinity of the metal film.
In the Lakowicz, et al. arrangement, the fluorescence response occurs at a very well defined beam incidence angle to the film, and the fluorescence signal passes to a detector through the glass. The drawback of Lakowicz, et al.'s system is that the fluorescence emerges as a cone of light on the backside of the optical arrangement making the detection geometry difficult and inflexible. The total internal reflection geometry used by Lakowicz, et al. and in various prior art sensors, makes them difficult to use in conventional fluorescence detection since the fluorescence does not emerge from the air (front) side of the device and further requires complex optics.
It would be desirable therefore to eliminate the deficiencies of the prior art fluorescence sensors by devising a fluorescence sensor compatible with commercial Fluorescence Optical Microscopes (FOMs).